These questions are "open" to me, not necessarily the scientific community. If you have insights or can point me toward references relevant to them, please do!

Open Questions

What causes an overloaded colony PCR reaction to fail?

How does plasmid copy number get affected by oxygen availability? Cellular media? I have noticed that LB often gives me orders of magnitude lower plasmid yields and that growing cultures with liquid:air volumes higher than 1:5 is not good.

How do I decide whether to gel purify after digestions (before ligations)? The BioBrick/Ginkgo manual doesn't instruct you to. Besty always does.